Dataset: 16S rRNA gene sequences of bacteria from members of an Arctic clade
Deployment: TN268

Culture media was prepared by pre-filtering seawater through a 0.8 µm polyethersulfone filter (Supor-200, Pall Corp, Ann Arbor, MI) and by sterilizing the filtrate using a 30 kD biomax polyethersulfone tangential flow filtration (TFF) cartridge (Millipore, Billerica, MA). TFF filter-sterilized seawater media was collected in autoclaved polycarbonate bottles and stored at in situ temperatures. Matching whole water samples were diluted (3-5 cells ml-1) in TFF filter-sterilized seawater media and added to each well of an acid washed (10% HCL) 96 well Teflon plate (Sonomatesting, Forestville, CA).

Each experiment consisted of 576 cultures divided into two treatments. One treatment contained filter sterilized seawater media (unamended) and one contained seawater media amended with a natural source of organic carbon (lysate). Vitamins B1, B6, B7, and B12 were added to the North Pacific gyre lysate treatment at a final concentration of 10 nM each. Plates were incubated in the dark at in situ temperatures (Puget Sound, 13 °C and North Pacific, 10 °C) and screened for growth on an Easyflow Guava flowcytometer equipped with a 96 well plate reader (Millipore, Billerica, MA). Cultures were checked for growth by transferring 150 μL of culture to a new plate and by staining the cells with Syber Green I (Invitrogen, Carlsbad, CA) diluted in TRIS buffer and at a final concentration of 1/2000, as previously described (Stingl et al., 2007).

Taxonomic assignments were determined for bacterial cultures that were positive for growth by extracting and amplifying the 16S rRNA gene. DNA from 200 µl of culture was extracted using a DNeasy Blood and Tissue Kit (QIAGEN, Germantown, MD, USA). 16S rRNA genes were amplified using a semi-nested PCR reaction with Taq polymerase (Fermentas, Hannover, MD, USA) and bacterial primers. Amplifications were performed in a C1000 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) using the following conditions: 35 cycles with 8F and 1492R primers followed by 38 cycles with 8F and 519R primers. The same conditions were used for each PCR reaction; denaturation at 94 °C for 30 s., annealing at 55 °C for one min, elongation at 72 °C for two min, and a final elongation step at 72°C for 10 min Amplicons were sequenced at the High-Throughput Genomics Unit (University of Washington, Seattle, WA, USA). Taxonomic assignments were determined using the Bayesian method of Wang et al., (2007) and a database augmented with sequences from marine environmental clades as previously described (Iverson et al., 2012).